The host response to infection by enteric pathogens is intestinal inflammation however many bacterial pathogens possess strategies to suppress this inflammatory response. Some enteric pathogens accomplish this by injecting bacterial effector molecules into the host cell cytoplasm via a syringe-like type III secretory system (T3SS). Enteropathogenic Escherichia coli (EPEC) is a foodborne non-invasive pathogen that causes diarrhea. EPEC virulence factors include a T3SS and secreted effector molecules. The hypothesis of this proposal is that the pro-inflammatory response of host cells to EPEC is driven by extracellular bacterial factors and that suppression of this host response depends on delivery of anti-inflammatory bacterial molecules into host cells by the T3SS. Compelling preliminary data suggest that the non-LEE encoded EPEC effectors, NleH1 and NleH2 (NleH1/2), possess anti-inflammatory activity and inhibit two pro-inflammatory signaling pathways, NF-:B and MAP kinase (MAPK). Additional preliminary data suggest that suppression of inflammation promotes colonization of the pathogen The overall goal of this proposal is to define the role of NleH1 and NleH2 in EPEC-induced anti-inflammatory activity, elucidate the underlying mechanism(s), and explore the role of these proteins in EPEC pathogenesis. This work is relevant to the VA healthcare mission as our troops, especially those stationed in remote areas overseas, are exposed to multiple enteric pathogens that cause significant morbidity thus reducing productivity. In addition, military recruits who have suffered infectious enteritis are at increased risk for the development of inflammatory bowel disease, especially Crohn's disease. Furthermore, defining the strategies by which bacterial pathogens modulate the host inflammatory response provides opportunities for the development of novel therapeutic agents for other intestinal inflammatory diseases. The following Specific Aims will address this hypothesis: Specific Aim 1. To define the roles of NleH1 and NleH2 in EPEC-induced anti-inflammatory activity. Specific Aim 2. To define the mechanism(s) by which NleH1/2 inhibit host inflammatory responses. Specific Aim 3. To determine the role of NleH1/2 in EPEC pathogenesis in a murine model of infection. Two independent in vitro models, infection and transfection, and an in vivo murine model of EPEC infection will be used to address these aims. Human and mouse intestinal epithelial cells will be infected with wildtype EPEC or deletion mutants nleH1, nleH2, or nleH1/2 and the effects on NF-:B and MAPK activation and on the expression of inflammatory cytokines will be tested. Cells will also be transfected to express bacterial proteins, NleH1, NleH2, or both, and then challenged with host cytokines, bacterial pro-inflammatory molecules, or live pathogens and the impact on activation of host signaling and production of inflammatory proteins will be analyzed. The in vitro findings will be correlated in an in vivo murine model of EPEC infection. Animals will be infected with wildtype EPEC or deletion mutant strains nleH1, nleH2, or nleH1/2, and the effects on intestinal inflammation and signaling and bacterial colonization will be determined. Additional mechanistic studies will be performed to identify specific amino acid residues or motifs in NleH1 and NleH2 that are responsible for the anti-inflammatory activity. The long-term goal of this proposal is to identify specific anti-inflammatory bacterial effectors and their mechanism(s) of action that would guide the development of therapeutic EPEC strains that attach to host intestinal epithelial cells and inject anti-inflammatory proteins but not interfere with other epithelial cell functions. PUBLIC HEALTH RELEVANCE: Infection with food- and water-borne pathogens increased 93% between 1979 and 2004 and deaths from GI infections tripled recently)(1). Infectious diarrhea diminishes significantly productivity especially of military personnel serving in resource-deficient countries. In addition, active duty military personnel who experienced infectious gastroenteritis had an increased risk (odds ratio, 1.4) for the development of inflammatory bowel disease (2). The mechanisms of pathogenesis of many of these infectious organisms are not understood. Studies focused on delineating how these bacteria cause disease will result in the development of new strategies for prevention, treatment, and avoidance of the sequelae that result from these infections. This research will focus on enteropathogenic E. coli (EPEC) and how these bacteria control the inflammatory response of the host by injecting proteins into intestinal cells through a needle-like structure, which block inflammation allowing the bacteria to persist for a longer time in the intestine.